The Plant Cell Wall
The plant cell wall is composed of independent, interacting networks of cellulose microfibrils tethered by hemicellulosic polysaccharides, which are embedded in a matrix of pectins, glycoproteins, and phenolic substances (Carpita and Gibeaut 1-30). The plant cell wall is not an immutable substance; rather, it is a self-(re)organizing barrier able to respond to external and internal stimuli to govern cellular defense, growth, and development. For a size perspective, plant cell walls are approximately 0.1-10 μm thick depending on the type of plant species. In comparison, the plasma membranes of plant cells are not more than 0.01 μm thick (Stephen C. Fry).
Plant cell walls are generally subdivided into two types: the primary cell wall and the secondary cell wall. Primary cell walls are synthesized and modified during a period of active cell growth and are often referred to as “growing cell walls” (Kerr and Bailey 327-49). Secondary cell walls are deposited after cell growth has stopped. Since cell wall material is deposited from the inside out, the secondary cell wall is interior to the primary cell wall. Although the bulk of mature plant cell walls are composed of secondary wall material, there is great interest in the components of the primary cell wall, and their controlled assembly and modification, because primary cell walls dictate cell type, shape, and size, and provide a selective barrier to the external environment.
The Hydroxyproline-Rich Glycoproteins
The hydroxyproline-rich glycoproteins (HRGPs), which include the extensins, proline-rich proteins, and arabinogalactan-proteins, contribute to the extracellular matrix throughout the plant kingdom and the Chlorophycean green algae (Kieliszewski and Lamport 157-72; Showalter and Varner 485-520). HRGPs are involved in all aspects of plant growth and development involving wall architecture (Goodenough et al. 405-17; Roberts 129-46) and wall assembly during embryogenesis (Hall and Cannon 1161-72) as well as responses to biotic and abiotic stress (Merkouropoulos and Shirsat 356-66; Merkouropoulos, Barnett, and Shirsat 212-19; Yoshiba et al. 115-22) that include mechanical stress (Shirsat et al. 618-24); (Hirsinger et al. 343-55), physical wounding (Chen and Varner 2145-51; Han et al. 59-70; Showalter et al. 547-65; Zhou, Rumeau, and Showalter 5-17), pathogenesis (Benhamou et al. 457-67), and symbiosis (Cassab 441-46; Franssen et al. 4495-99; Frueauf et al. 429-38).
HRGPs are extended macromolecules consisting of small repetitive peptide and glycopeptide motifs that form peptide modules and glycomodules of functional significance, as in “mix-and-match” mode they define the molecular properties of the overall macromolecule (Kieliszewski et al. 538-47; Kieliszewski and Lamport 157-72). The glycomodules result from a combination of posttranslational modifications unique to plants, namely proline hydroxylation (Lamport 1438-40) and its subsequent glycosylation (Lamport 1322-24) that leads either to short arabinooligosaccharide or larger arabinogalactan polysaccharide addition to the Hyp residues. A sequence-dependent O-Hyp glycosylation code directs the precise addition of oligosaccharides and polysaccharides (Kieliszewski 319-23) and there is increasing evidence that other sequence-dependent codes direct inter- and intramolecular crosslinking of HRGPs. Crosslinked HRGPs, including extensins, contribute to wall architecture and defense responses by forming interpenetrating crosslinked networks in the wall. However, the precise identity of the intermolecular crosslink(s) has remained elusive.
The Extensins
Extensins are structural HRGPs that are covalently crosslinked into the primary cell wall, rendering them insoluble. Aside from being rich in Hyp, they also are rich in Ser, Tyr, Lys, Val and Thr. They are extensively post-translationally modified with short Hyp-O-oligoarabinosides and typically possess monogalactosyl-serine (Lamport, Katona, and Roerig 125-31; Lamport and Miller 454-56). Extensins are rigid linear molecules that adopt a polyproline II left-handed helical conformation (3 residues per turn, 9.4 Å pitch) (van Holst and Varner 247-51); (Heckman, Terhune, and Lamport 848-56); (Stafstrom and Staehelin 242-46).
Three major types of extensin precursors to the extensin network are widespread in dicot plants, namely Precursor 1 (P1) extensins that are characterized by the repetitive motif: Ser-Hyp-Hyp-Hyp-Hyp-Thr-Hyp-Val-Tyr-Lys (SEQ ID NO: 1), (Smith et al. 1021-30; Smith, Muldoon, and Lamport 1233-39), P2 extensins that contain repeats of the motif Ser-Hyp-Hyp-Hyp-Hyp-Val-Tyr-Lys-Tyr-Lys (SEQ ID NO: 2) (Smith et al. 1021-30; Smith, Muldoon, and Lamport 1233-39), and finally, the P3 extensins that contain a major palindromic (bolded) repeat: Ser-Hyp-Hyp-Hyp-Hyp-Ser-Hyp-Ser-Hyp-Hyp-Hyp-Hyp-Tyr-Tyr-Tyr-Lys (SEQ ID NO: 3) (Lamport 79-115; Smith et al. 1021-30).
The P1 and P2 extensins can be isolated by the salt-elution of intact cells as soluble monomer precursors to the extensin network (Fong et al. 548-52; Smith et al. 1021-30; Smith, Muldoon, and Lamport 1233-39). The P3 extensins, on the other hand, have never been isolated as monomeric precursors to the extensin network, presumably due to their rapid incorporation into the cell wall via covalent intermolecular crosslinking (Mort and Lamport 289-309). Consequently, the molecular properties of soluble monomeric P3 extensins have thus far been inferred from gene sequences, and P3 glycopeptides and peptides rendered from cell walls (Corbin, Sauer, and Lamb 4337-44; Lamport 1155-63; Lamport 27-31; Lamport 79-115; Lamport and Miller 454-56; Showalter et al. 547-65; Showalter and Varner 375-92; Zhou, Rumeau, and Showalter 5-17).
Intramolecular Extensin Crosslinking
Both P2 and P3 type extensins can undergo intramolecular crosslinking of Tyr residues (Epstein and Lamport 1241-46) (underlined in the sequences above) to form the diphenylether crosslink amino acid, isodityrosine (IDT) (FIG. 1) (Fry 449-55). First observed as an unknown tyrosine derivative in extensin peptides (Lamport 1155-63), and later identified in wall hydrolysates (Fry 449-55), and also as a component of trityrosine in Ascaris cuticle collagen (Fujimoto 637-43), IDT was initially hypothesized to be an intermolecular crosslink responsible for transforming the soluble extensin monomeric precursors, P1, P2 and P3, into an insoluble extensin network in muro (Fry 449-55); (Lamport and Epstein 73-83). But IDT was identified only as an intramolecular crosslink in P2 and P3-derived extensin peptides purified from enzymic digests of cell walls (Epstein and Lamport 1241-46) and extensin peptides crosslinked by intermolecular IDT have never been isolated. Although it has been suggested that extracellular peroxidases catalyze the formation of IDT, to date, the precise mechanism in muro remains a mystery (Fry 853-62).
Intermolecular Extensin Crosslinking
Evidence has suggested that pectin-protein crosslinks may play a role in extensin insolublization in muro (Keegstra et al. 188-96; Mort; Qi et al. 1691-701). Although these crosslinks may exist to some extent, the extensin network in cell walls and in vitro remains insoluble after complete deglycosylation with anhydrous hydrogen fluoride at 0° C. (Mort and Lamport 289-309; Schnabelrauch et al. 477-89). Furthermore, Hyp-rich extensin peptides are only released after proteolytic cleavage of a partially or fully deglycosylated extensin network (Lamport 1155-63; Lamport 79-115; Mort and Lamport 289-309; Qi et al. 1691-701). Thus, the insolubility of cell wall extensins is primarily attributed to a protein-protein and/or protein-phenolic-protein crosslink(s) (Mort and Lamport 289-309).
Peroxidase-catalyzed extensin intermolecular crosslinking has been demonstrated in vitro by several groups (Brownleader et al. 1115-23; Everdeen et al. 616-21; Jackson et al. 1065-76; Price et al. 41389-99; Schnabelrauch et al. 477-89), yet the molecular nature of the intermolecular crosslink(s) has not been were not identified. Indeed, Lamport and colleagues (Schnabelrauch et al. 477-89) found no IDT increase in P1 extensins after their crosslinkage in vitro by a tomato pl 4.6 extensin peroxidase, although the abundance of Val-Tyr-Lys motifs in several crosslinking extensins, including P1 and P2, suggested the intermolecular crosslinks involved tyrosine and/or lysine (Schnabelrauch et al. 477-89).
More recently, Brady and Fry identified a trimeric tyrosine derivative, pulcherosine (FIG. 2) and the tetrameric tyrosine derivative, di-isodityrosine (dilDT; FIG. 3) (Brady, Sadler, and Fry 349-53); (Brady, Sadler, and Fry 323-27) in tomato cell wall hydrolysates and speculated that IDT-containing extensins could be insolubilized through intermolecular IDT crosslinks forming pulcherosine and di-isodityrosine (FIG. 4). Also, as extensin incorporation into the cell wall increased, hydrolysates of these walls showed that the amounts of IDT decreased and dilDT increased (Brady and Fry 87-92). These findings suggest a significant role for IDT-rich extensins, which are supported by the recent discovery that RSH, an extensin containing 14 intramolecular IDT motifs, is crucially involved in positioning the cell plate during the earliest stages of embryogenesis in Arabidopsis (Hall and Cannon 1161-72).
However, to date, there has been no direct demonstration of an extensin intermolecular crosslink that involves either Tyr or Lys. The results of in vitro assays have been difficult to interpret, as the substrate extensins, P1 and P2, contain both Tyr and Lys and the amino acids formed by crosslinking were not identified (Brownleader et al. 1115-23; Everdeen et al. 616-21; Fujimoto 637-43; Hall and Cannon 1161-72; Schnabelrauch et al. 477-89). Other approaches involving the isolation of intermolecularly crosslinked peptides from the cell wall itself have also proven intractable (Epstein and Lamport 1241-46).